1. Field of the Invention
The present invention relates to a labeling means for protein by selenomethionine utilizing a wheat embryo cell-free protein synthesis system and also to a labeling means for protein using heavy hydrogen.
2. Description of the Related Art
As the completion of the genome project is coming near, focus of the research task has been quickly developing from analysis of gene structures to analysis of gene functions. It is presumed that intracellular protein does not function solely but its function comes into effect as a result of interaction with various protein factors, nucleic acids, lower molecular species, cell membrane components, etc. in a cooperative manner and further that a biological function takes place as a total sum of the said interaction. One of the focuses of the research task after the genome project is to analyze the relation between function of various kinds of protein factor complexes and its structure. Fruit obtained therefrom is expected from research of basic biology, etc. including structural biology and biochemistry to clarify the relation between etiology and gene translation product in a medical field as its application and also to provide extremely important findings in developments of pharmaceuticals.
In the structural biological research of protein, there have been used X-ray crystal analysis method, NMR spectroscopy and neutron scattering method. In order to carry out those methods, it is essential to prepare a large quantity of protein which is labeled with heavy atom, isotope and heavy hydrogen, respectively and maintains the activity. In the conventional X-ray crystal analysis method, it is necessary to prepare at least two kinds of crystals, i.e. “crystal of protein of a natural type” and “heavy atom-labeled crystal of protein where is substituted by heavy atom” for the determination of lattice constant. It is often difficult to prepare the latter crystal and, therefore, there are many cases where crystal structure is not clarified in spite of the fact that the crystallization of the former has been successful. With regard to a technique which has been proposed as a solving means therefor and partly utilized, there is a method utilizing selenomethionine where sulfur atom of methionine is substituted with selenium which is a heavy atom. That is a method in which aimed gene is introduced into incubated cells or Escherichia coli followed by incubating in a medium containing selenomethionine. This is a genetic technological means for expressing “the protein for which the gene codes” whereby selenomethionine is introduced into the said protein.
On the other hand, it is necessary to substitute hydrogen atom of protein with heavy hydrogen for steric structure analysis of protein by an NMR spectroscopy or a neutron scattering method and locality analysis of H2O molecule in a molecule. With regard to a method therefor, there has been used a means where aimed gene is introduced into microbes such as Escherichia coli and is incubated in a medium containing an amino acid labeled with heavy hydrogen whereupon genetic product is expressed and protein labeled with heavy hydrogen is prepared.
Selenomethionine-containing protein is useful since it is a one-type crystal obtained from the said protein and its structural analysis is possible and that has been proved already. However, there are many disadvantages in the conventionally conducted method for the labeling of protein with selenomethionine. They are (1) methionine is an essential amino acid which participates not only in protein synthesis but also in various metabolisms in cells and, in addition, since selenium itself shows a strong cytotoxicity, it is not possible to substitute all methionine in the medium with selenomethionine whereby incubation in large quantities is necessary for the preparation of selenomethionine-containing protein in a required amount and (2) selenomethionine is expensive and also highly toxic and, therefore, there is a big problem for a method of discarding the medium containing high concentrations of selenomethionine after the incubation.
On the other hand, labeling of protein with heavy hydrogen is an essential condition for steric structure analysis of protein by an NMR spectroscopy or by a neutron scattering method but, until now, an incubation method of microbe as mentioned above has been an only one. In that method, there are many disadvantages which are to be solved that 1) operation is troublesome, 2) labeling efficiency is low and 3) cost is very high because of necessity of large quantities of amino acid labeled with heavy hydrogen and, in addition, there is a serious problem for a method of discarding the medium due to its strong toxicity.
Further, due to the same reason, there has been developed no practical means using genetic engineering means for the preparation of protein containing amino acid which is labeled with carbon 13 (13C) or nitrogen 15 (15N) to be used in an NMR spectroscopy.
Under such current circumstances, there has been a brisk demand for the means of preparing a labeled protein which has good efficiency, is less expensive and shows little amount to be discarded as liquid in which the desired amino acid in an aimed protein molecule is able to be labeled in a desired manner where the activity of the said protein is still maintained.